| Candida infections have become a serious medical problem due to high incidence and mortality in AIDS patients, transplant recipients and other immunosuppressed individuals. Despite continuous expansion in the arsenal of antifungal drugs, antifungal drugs available cannot meet the increasing requirements for managing infections in medically complex patients.2-aminotetralin derivates were synthesized as novel chemical structural antifungal agents. Of them,10b was found to have the strongest antifungal activity in vitro. The aim of the present work was to further investigate the antifungal activity of 10b as well as its mechanism of action in vitro. Broth microdilution analytic results showed that 10b possessed potent activities against almost all C. albicans isolates, which was similar to or more powerful than that of ketoconazole (KCZ) or fluconazole (FLC) and MIC80 value ranged from 0.063 to 8μg/ml. Despite high resistance to FLC and ICZ (both MIC80 value 256μg/ml) for isolate SC5314R,10b displayed powerful antifungal activity against this strain, MICgo value being 0.125μg/ml. Agar disk diffusion test further confirmed that 10b possesses more powerful antifungal activity against FLC-resistant isolates than that of FLC and KCZ and exhibits a fungicidal activity to some extent. Growth curve test, Half Inhibitory Concentration (IC50) and Minimal Fungicidal Concentration (MFC) determination also confirmed the potent antifungal activity of 10b. In addition, crystal violet staining method and XTT reduction assay were performed to investigate the anti-biofilm activity of 10b. The analytic results showed 10b dramatically inhibit biofilm formation and siginificantly reduced mature (48 h) biofilm metabolic activity. Moreover, the inhibitory effect of 10b was more powerful than that of farnesole. Confocal laser scanning microscopy (CLSM) showed that preincubated with 0.1μM 10b, the typical architecture of biofilms (intertwining mycelial structures and a basal layer of blastospores) was destructed although pseudohyphae and true hyphae were observed. When the concentration was increased by 10-fold, the adherent yeast cells were successfully prevented from germination and resulted in scant or nonexistent biofilms. To clarify the underlying action mechanism, Thin-Layer Chromatography (TLC) assay and Gas Chromatograph-Mass Spectrometer (GC/MS) assay were performed to investigate the targets of 10b in the ergosterol biosynthetic pathway amd transmission electron micrograph was used to observe the ultrastructure of C. albicans cells. Then, cDNA microarray study and realtime RT-PCR assay were conducted to study and research the genes expression profiles of yeast C. albicans or C. albicans biofilms treated or untreated with 10b. Finally, cytotoxicity of 10b in vitro was assessed by MTS/PMS reduction assay. TLC analytic results showed that sterols in C. albicans strain treated by 10b were evidently different from that of treated by KCZ. Their major differences in sterol classes were most pronounced in lanosterol and its analogues. The spottednesses of lanosterol and its analogues in KCZ-grown cells were significantly bigger and deeper than that of control-grown cells while disappeared in 10b-grown cells. It is presumed that 10b possesses a mode of action different from KCZ in ergosterol biosynthetic pathway.10b might inhibit the activities of ergosteol metabolic enzymes except for lanosterol 14a-demethylase and resulted in lanosterol changing into other intermediate sterols. GC/MS analytic results showed that 10b produced a profile similar to that of homozygous erg24 strain, in which ignosterol and ergosta-8,14,22-trienol were major sterols and no trace of 24-methylene-lanost-8-en-3-ol was found. However, KCZ-grown cells had a profile with 24-methylene-lanost-8-en-3-ol like the predominant sterol. We therefore postulate that sterol C-14-reductase, encoded by ERG24, instead of lanosterol 14α-demethylase, encoded by ERG11, is an important target for 10b in this pathway.10b also showed an action mechanism different from that of morpholines in the ergosterol biosynthetic pathway, although both of them are sterol C-14-reductase inhibitors. Morpholines inhibit sterol C-8 isomerase, encoded by ERG2 gene, resulting in ergosta-8-en-3-ol accumulation. As sterol C-8 isomerase is situated in the downstream of sterol C-14-reductase, Fm-grown erg24 strain produced a profile completely identical to that of erg24 strain. Although the sterol composition of 10b-grown cells was completely identical to that of erg24 strain, the ratio of ergosta-8,14, 22-trienol to ignosterol was different. The content of ergosta-8,14,22-trienol in 10b-treated erg24 strain was much more than that of in erg24 strain without 10b treatment, indicating that there must be another important target upstream sterol C-14 reductase. We deduced that another target was an enzyme related to sterol C-5 desaturase, which catalyzes the conversion of ergosta-8,14,22-trienol to ergosta-5,8,14,22-tetraenol. In addition, we found that the content of ignosterol built up in the wake of increase in the concentration of 10b, indicating that sterol C-5 desaturase related enzyme was the dominant target of 10b at a low concentration, while the affinity of 10b with sterol C-14 reducase augmented along with the increase in the concentration of 10b and finally changed into the fundamental target at a higher concentration. Although the ERG24 gene, encoding the C-14 sterol reductase, has been reported to be essential to the aerobic growth of Saccharomyces cerevisiae, the erg24 mutant of C. albicans is capable of growth under normal aerobic conditions on standard defined and enriched media. Transmission electron micrographs show that 10b treatment cannot changes the integration and permeability of the fungal membrane. This indicates that the major mechanism of action of 10b against C. albicans is uncorrelated to inhibiting the activity of sterol C-14 reducase in ergosterol biosynthetic pathway. Then a cDNA microarray study and real-time RT-PCR assay were performed to further clarify the mechanism of action of 10b. Yeast C. albicans cDNA microarray and realtime RT-PCR analytic results showed that 10b treatment resulted in marked down-regulation in metabolism-related genes, including glycolysis-related genes (e.g., PFK1, CDC19 and HXK2), fermentation-related genes (e.g., PDC11, ALD5 and ADH1) and respiratory electron transport chain-related genes (e.g., CBP3, COR1 and QCR8). Real-time fluorimetric assay revealed that 10b treatment increased generation of endogenous reactive oxygen species (ROS), which was similar to or more powerful than that of miconazole (MCZ). Function analytic results shawed that 10b treatment decreased mitochondrial membrane potential (ΔΨm), ubiquinone-cytochrome C reductase (complex III) activity and intracellular ATP level in C. albicans SC5314 strain. Besides, addition of antioxidant ascorbic acid (AA) reduced the antifungal activity of 10b significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation might contribute to the antifungal activity of 10b against yeast C. albicans. C. albicans biofilm cDNA microarray and realtime RT-PCR results showed that 10b treatment resulted in a striking down-regulation of hypha-specific gene ECE1 and a marked up-regulation of transcriptional repressors NRG1 in C. albicans biofilms, which is directly linked to inhibit biofilm formation. Moreover, the expressions of glycolysis related genes (e.g., HXK2 and PFK1), fermentation-related genes (e.g., ADH1) and antioxidant defense (e.g., SOD5) were decreased markedly. Functional analysis indicated that addition of anti-oxidant ascorbic acid reduced inhibitory efficiency of 10b on mature biofilm. These results indicated that inhibition of 10b on biofilm formation possibly depends on impairing the ability of C. albicans to change its morphology via altering the expression of biofilm formation genes. Mitochondrial aerobic respiration shift and endogenous ROS augmentation might be a major contribution to reduce mature biofilm metabolic activity. Although 10b seemed to reduce MTS/PMS reduction in a dose dependent manner, IC50 value for mammalian cells was much higher than MIC50 value for C. albicdns. This indicates that the formulation is preliminarily safe and warrants further study for possible human applications. Our data provide useful information for the development of new antifungal agents with novel chemical structure and distinct targets in ergosterol biosynthetic pathway and of new strategies to reduce candida infections.
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